The mammalian dentition is a premier model for understanding how organs form. In particular, it exemplifies the recursive and widely deployed developmental strategy of inductive epithelial-mesenchymal (E-M) tissue interactions, by which signals are sequentially and reciprocally exchanged between interacting epithelium and mesenchyme, resulting in the progressive differentiation of each tissue. In the prior grant period, we developed important new methods for reconstructing the gene regulatory network (GRN) that underlies early molar tooth formation, and we identified a unique E-M Wnt-Bmp4 feedback circuit that controls key steps in this process. In addition to providing a mechanism for the pivotal roles of Bmp and Wnt in odontogenesis, this Wnt-Bmp4 feedback circuit can explain several specific properties of E-M organogenesis, including phasic E-M expression dynamics and the coupling of E-M development. Thus, the reconstruction of this odontogenic GRN affords a systems level understanding of how vertebrate organs form. We hypothesize that this GRN can be further developed to enable the efficient manipulation of organ fate, with profound significance for regenerative medicine. We will test this hypothesis by pursuing three Specific Aims. In Aim 1, we will significantly improve the version 1.0 odontogenic GRN that controls early murine molar tooth development from initiation- to cap-stage. We will obtain new gene expression data from unique fluorescently labeled dental epithelial and mesenchymal progenitor cell populations, compare these profiles to our existing datasets, and use this data to add a spatial resolution parameter to the odontogenic GRN. We will also obtain microRNA data via RNA-Seq, functionally validate selected miRNAs, and integrate critical miRNA regulators into the GRN. In Aim 2, we will perform DNase-Seq and ChIP-Seq analysis on uninduced and induced dental mesenchyme, using ChIP-Seq for histone modifications (H3K4me1, H3K4me3, H3K27me3, H3K27ac), co-activator marks (p300), and specific TFs that occupy key nodal points in the existing odontogenic GRN (e.g., Msx1, Pax9). We will also assay epigenetic marks in Msx1 and Hdac3 mutant mice. New bioinformatic methods will be developed to integrate this data into the existing GRN. Lastly, in Aim 3, we will validate the new version 2.0 GRN both in vitro and in vivo. In vitro, we will perturb tooth germ explant cultures and primary dental mesenchymal cells in defined ways, and we will compare the gene expression responses to those observed in endogenous dental tissues. We will also seek to validate the odontogenic GRN in vivo, by activating odontogenesis in the mouse oral cavity genetically and via treatment with specific factors identified from the GRN. These experiments hold the potential to provide the most complete GRN for any vertebrate organ, and will identify key nodal points for intervention. Ideally, this work will provide an instructional template for defined organ replacement.